Sophorolipids as protein inducers and inhibitors in fermentation medium

ABSTRACT

A method for producing sophorolipids having protein inducer and/or repressor activities having the steps of synthesizing the sophorolipid by fermentation of  Candida bombicola  in a fermentation media to form a natural mixture of lactonic sophorolipids and non-lactonic sophorolipids and then utilizing the natural mixture as a protein inducing agent, utilizing the natural mixture as a protein repressing agent, and/or utilizing the natural mixture as a combined protein induction/repressor agent. An application of the sophorolipid compound produced according to the method as a microbial media component.

STATEMENT OF RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 11/722,356 having a filing date of 11 Dec. 2007, which is the USNational Phase application of International Application No.PCT/US2005/046457 having an International Filing Date of 22 Dec. 2005,which is based on and claims priority on U.S. Provisional PatentApplication No. 60/638,747 having a filing date of 22 Dec. 2004.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention in general relates to the field of uses forsophorolipids and more specifically to the field of protein inducers andas a media component. The invention is directed to developing low costinducers/repressors for protein production.

2. Prior Art

For production of proteins on a larger scale, a need exists for cheapand well defined molecules to act as inducers of the protein of interestand also as a repressor for unwanted enzymes. This aids in higheryields, decreasing the purification costs and enhancing the profits.Sophorose has been shown to be a good inducer of cellulase protein. SeeHrmova, M., Petrakova, E., Biely, P., Journal of General Microbiology137, 541-547 (1991). However, the cost of the molecule makes itimpractical to be used at commercial level.

Sophorolipids are microbial extracellular glycolipids produced byresting cells of Candida bombicola. The chemical composition ofsophorolipid is constituted by a disaccharide sugar viz. sophorose and afatty acid or an ester group. Candida bombicola produces thesophorolipids as a mixture of macroloctones and free acid structuresthat are acetylated to various extents at the primary hydroxyl sophorosering positions (FIG. 1). See Bisht, K. S. et al., J. Org. Chem., vol.64, pp. 780-789 (1999).

It is to the development of sophorolipids for production and industrialpurposes, and other purposes, that the present invention is directed.

BRIEF SUMMARY OF THE INVENTION

A natural mixture of sophorolipids was synthesized by fermentation ofCandida bombicola. When B. subtilis was grown in presence ofsophorolipids, increased production of amylase was observed. WhenPleurotus ostreatus 473 strain, which produces both laccase andmanganese peroxidase, was grown in the presence of sophorolipids,increased production of laccase was observed while the production ofmanganese peroxidase decreased.

Thus, the applications of sophorolipids in the fields of industry andbasic sciences would have tremendous advantages in higher production orinhibition rates of proteins. Further, while sophorolipids can findapplications in diversified fields, the present invention indicates thatsophorolipids could be used as protein inducers and repressors.

These features, and other features and advantages of the presentinvention, will become more apparent to those of ordinary skill in therelevant art when the following detailed description of the preferredembodiments is read in conjunction with the appended drawing.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 are representative structures of sophorolipids produced byCandida bombicola, with FIG. 1A showing lactonic sophorolipid and FIG.1B showing open ring sophorolipid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. Sophorolipid Fermentation

Sophorolipids were synthesized by fermentation of Candida bombicola. Thefermentation media was composed of glucose 100 g, yeast extract 10 g,urea 1 g and oleic acid 40 g in 1000 ml of water. After 7 days offermentation, sophorolipid was extracted thrice using ethyl acetate. Theextracts were pooled and the solvent then was removed. The obtainedproduct was therein washed with hexane to remove the residual fattyacids. This was “natural” sophorolipid. The sophorolipid was dried in avacuum desiccator.

2. Study of Amylase Protein Induction

Bacillus subtilis strain was used to study the effect of sophorolipid onamylase induction. Culture was grown on Peptone: 5 gm/L, Beef extract: 3gm/L and Starch: 2 gm/L. A single colony of Bacillus subtilis wasinoculated in 10 ml medium and was allowed to grow for 12 hours. Afterthe culture reached to the log phase, 1 ml of the same was inoculated in100 ml of the medium. Control experiments having no sophorolipids alsowere conducted, whereas the experimental set has 1 mM of sophorolipidprepared in DMSO.

After 48 hours a sample was withdrawn, centrifuged at 8000 rpm for 20minutes, and amylase activity determined using standard methods.

3. Study of Laccase Protein Induction and Manganese Peroxide Repression

Pleurotus ostreatus 473 was used to study the effect of sophorolipids onLaccase/Manganese peroxidase production. Culture was cultivated in agarmedium containing 5 g/L cellulose, 1 g/L KH₂PO₄, 0.5 g/L MgSO₄.7H₂O, 0.2g/L ammonium tartrate, 0.2 g/L NaH₂PO₄, 50 mg/L CaCl₂, 50 mg/L,FeSO₄.7H₂O, 10 mg/L CuSO₄. 5H₂O, 5 mg/L ZnSO₄.7H₂O, 5 mg/L MnSO₄.4H₂Oand 25 g/L agar. pH of the medium was 6.0. For enzyme productionstudies, flasks with 20 mL of the above media (without agar) wereinoculated with two wort agar plugs (2° Balling, 10 mm diameter), cutfrom the actively growing part of a colony on a Petri dish, andincubated at 27° C. for 14 days. After 14 days a sample was withdrawn,centrifuged at 8000 rpm for 20 minutes and laccase and MnP activitydetermined using standard methods.

RESULTS AND DISCUSSION

As shown in Table 1, upon growing B. subtilis in the presence ofsophorolipid, an increase of 39% amylase protein production observed.Growing Pleurotus ostreatus 473 in the presence of 1 mM sophorolipidnatural mixture resulted in 4.5 times increase in laccase proteinwhereas there was a decrease of 25% in the production of manganeseperoxide protein. This shows that the sophorolipid natural mixture hasthe ability to act as an inducer for some proteins and the ability toact as a repressor for others.

The invention will find its place in industries that produce proteinproducts such as enzymes through microbial fermentation. Also, microbialmedia that has sophorolipids as a component can be sold for proteininduction studies.

Sophorolipids will be administered in the fermentation medium as eitherfine powder or in the form of solutions prepared in DMSO, alkalinesucrose solution, chloroform, methanol or ethyl acetate.

TABLE 1 % Increase/decrease of enzyme production Enzyme % values LaccaseControl 100 Sophorolipid Induced 453 Manganese peroxidase Control 100Sophorolipid Induced 73 Amylase Control 100 Sophorolipid induced 139

The foregoing detailed descriptions of the preferred embodiments and theappended FIGURE have been presented only for illustrative anddescriptive purposes and are not intended to be exhaustive or to limitthe scope and spirit of the invention. The embodiments were selected anddescribed to best explain the principles of the invention and itspractical applications. One skilled in the art will recognize that manyvariations can be made to the invention disclosed in this specificationwithout departing from the scope and spirit of the invention.

What is claimed is:
 1. A method for producing sophorolipids havingprotein inducer and/or repressor activities comprising the steps of: a)synthesizing the sophorolipid by fermentation of Candida bombicola in afermentation medium to form a natural mixture of lactonic sophorolipidsand non-lactonic sophorolipids; and b) utilizing the natural mixture asa protein inducing agent.
 2. The method according to claim 1, whereinthe fermentation medium comprises glucose, yeast extract, urea, andoleic acid in water.
 3. The method according to claim 2, furthercomprising, after a period of fermentation, extracting the sophorolipidusing ethyl acetate as a solvent.
 4. The method according to claim 3,further comprising removing the solvent and washing the obtained productwith hexane to remove residual fatty acids, resulting in the naturalmixture of sophorolipids.
 5. The method according to claim 4, furthercomprising drying the natural mixture of sophorolipids.
 6. The methodaccording to claim 1, further comprising adding an effective amount ofthe natural mixture of sophorolipids as a protein inducing agent to amicrobial culture medium.
 7. The method according to claim 6, whereinthe protein is selected from the group consisting of amylase, laccase,and manganese peroxidase, and combinations thereof.
 8. A method forproducing sophorolipids having protein inducer and/or repressoractivities comprising the steps of: a) synthesizing the sophorolipid byfermentation of Candida bombicola in a fermentation medium to form anatural mixture of lactonic sophorolipids and non-lactonicsophorolipids; and b) utilizing the natural mixture as a proteinrepressing agent.
 9. The method according to claim 8, wherein thefermentation medium comprises glucose, yeast extract, urea, and oleicacid in water.
 10. The method according to claim 9, further comprising,after a period of fermentation, extracting the sophorolipid using ethylacetate as a solvent.
 11. The method according to claim 10, furthercomprising removing the solvent and washing the obtained product withhexane to remove residual fatty acids, resulting in the natural mixtureof sophorolipids.
 12. The method according to claim 11, furthercomprising drying the natural mixture of sophorolipids.
 13. The methodaccording to claim 8, further comprising adding an effective amount ofthe natural mixture of sophorolipids as a protein repressing agent to amicrobial culture medium.
 14. The method according to claim 13, whereinthe protein is selected from the group consisting of amylase, laccase,and manganese peroxidase, and combinations thereof.
 15. A method forproducing sophorolipids having protein inducer and/or repressoractivities comprising the steps of: a) synthesizing the sophorolipid byfermentation of Candida bombicola in a fermentation medium to form anatural mixture of lactonic sophorolipids and non-lactonicsophorolipids; and b) utilizing the natural mixture as a combinedprotein induction/repressor agent.
 16. The method according to claim 15,wherein the fermentation medium comprises glucose, yeast extract, urea,and oleic acid in water.
 17. The method according to claim 16, furthercomprising, after a period of fermentation, extracting the sophorolipidusing ethyl acetate as a solvent.
 18. The method according to claim 17,further comprising removing the solvent and washing the obtained productwith hexane to remove residual fatty acids, resulting in the naturalmixture of sophorolipids.
 19. The method according to claim 18, furthercomprising drying the natural mixture of sophorolipids.
 20. The methodaccording to claim 15, further comprising adding an effective amount ofthe natural mixture of sophorolipids as a protein inducing andrepressing agent to a microbial culture medium.
 21. The method accordingto claim 20, wherein the protein is selected from the group consistingof amylase, laccase, and manganese peroxidase, and combinations thereof.